Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

ABSTRACT

The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

ACKNOWLEDGMENTS

This research was supported in part by the Florida Agricultural Experiment Station and by grants from the U.S. Department of Energy, Office of Basic Energy Research (FG05-86ER3574); the National Science Foundation (DMB 8204928); and the U.S. Department of Agriculture, Alcohol Fuels Program (86-CRCR-1-2134). We gratefully acknowledge the support of Y. A. Osman by the Egyptian Ministry of Higher Education.

We appreciate the excellent technical assistance of J. M. Neville during the construction and screening of M13 clones.

BACKGROUND OF THE INVENTION

In the obligately fermentative bacterium Zymomonas mobilis, the regeneration of NAD⁺ depends upon two enzymes, i.e., pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1)(27, 37) Z. mobilis is the only known obligately fermentative procaryote which utilizes an Entner-Doudoroff pathway for glycolysis and, like Saccharomyces cerevisiae, produces ethanol and carbon dioxide as dominant fermentation products. We recently reported the sequence of the pyruvate decarboxylase gene from Z mobilis (9) and have now cloned and sequenced the principal alcohol dehydrogenase isozyme from this organism, the adhB gene.

Z. mobilis contains two isozymes for alcohol dehydrogenase which are readily distinguished on acrylamide gel zymograms, by ion-exchange chromatography (18, 39), and by their ability to oxidize butanol (20, 28). Although alcohol dehydrogenase II is the most abundant isozyme, both appear to contribute during fermentation (20, 28). Both isozymes have been purified, and the N-terminal amino acid sequences have been determined by polypeptide sequencing (28). The alcohol dehydrogenase I isozyme from Z. mobilis is a tetramer containing zinc with a monomeric molecular weight of between 34,000 and 40,000. The alcohol dehydrogenase II isozyme from Z. mobilis is reported to be somewhat smaller, a tetramer with a monomeric molecular weight between 31,000 and 38,000 (18, 20, 28, 36, 39). This isozyme is quite unusual, containing iron rather than zinc in its active site (28, 36). The alcohol dehydrogenase II isozyme is the most abundant alcohol dehydrogenase activity in Z. mobilis and has a high degree of specificity for ethanol as a substrate (20, 28, 39).

In this paper, we describe a novel indicator plate method for the direct detection of clones which express alcohol dehydrogenase. This technique has been used to clone the gene encoding alcohol dehydrogenase II (adhB) from Z. mobilis. This gene was sequenced, and the sites for transcriptional initiation were determined.

The invention of the subject application was disclosed in an article published in the Journal of Bacteriology, June, 1987, pp. 2591-2597.

BRIEF SUMMARY OF THE INVENTION

The gene which encodes alcohol dehydrogenase II (adhB) from Zymomonas mobilis was cloned in Escherichia coli as a 1.4-kilobase DNA fragment by using a novel indicator plate which directly detects the expression of this activity by recombinant colonies. The DNA sequence for this clone contained an open reading frame encoding a polypeptide of 383 amino acids, with a molecular weight of 40,141. Although this protein exhibited very little homology with other known alcohol dehydrogenases, the predicted amino acid composition was in excellent agreement with that reported for the purified alcohol dehydrogenase II protein from Z. mobilis. In Z. mobilis, the adhB gene was transcribed from tandem promoters which were separated by 100 base pairs and ended with a transcriptional terminator (13-base-pair palindrome). In Escherichia coli, only one of the Z. mobilis promoters was used, despite apparent similarity to the enteric consensus promoter. The adhB gene was transcribed at low levels in E. coli from the P2 promoter of Z. mobilis but was expressed well in E. coli under control of the lac promoter (approximately 0.25% of the total cell protein).

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods

Bacterial strains, plasmids, and growth conditions. The strains and plasmids used in this study are described in Table 1. Z. mobilis CP4 was grown in complex medium (29) at 30° C. without agitation. E. coli strains were grown at 37° C. in Luria broth (24) containing added carbohydrate only when indicated below. Solid media contained agar (15 g/liter). Transformants of E. coli were isolated and identified by using media containing ampicillin (50 mg/liter), chloramphenicol (40 mg/liter), and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside; 20 mg/liter) and by using an aldehyde indicator plate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows positive colonies detected after 20 minutes to 2 hours at 37° C.

FIG. 1B shows negative colonies surrounded by a halo.

FIGS. 2-1, 2-2, 2-3, 2-4 and 2-5 is the DNA sequence and translated amino acid sequence of adhB from Z. mobilis.

FIG. 3A shows comparisons to sequencing ladder from pLOI286.

FIG. 3B shows comparisons to sequencing ladder from pLOI284.

Aldehyde indicator plates for the direct detection of clones producing alcohol dehydrogenase. Mixtures of pararosaniline and bisulfite are often referred to as Schiff reagent and have been widely used to detect aldehydes (22), to detect sugars on glycoproteins after periodic acid oxidation (14, 16), and in a broth test for organisms such as Z. mobilis which secrete aldehydes (11). These components have been incorporated into a solid medium which is relatively nontoxic and can be used to identify clones expressing enzymes which produce aldehydes.

Indicator plates were prepared by adding 8 ml of pararosaniline (2.5 mg/ml of 95% ethanol; not autoclaved) and 100 mg of sodium bisulfite (unsterilized dry powder) to 400-ml batches of precooled (45° C.) Luria agar lacking added carbohydrate. Most of the dye was immediately converted to the leuco form by reaction with the bisulfite (22) to produce a rose-colored medium which was dispensed into petri plates. Plates were stored at room temperature, away from fumes which contain aldehydes (cigarette smoke, many plastic containers, etc.) and light, both of which promote increased background color.

Ethanol diffuses into the E. coli cells, where it can be converted to acetaldehyde by alcohol dehydrogenase. The leuco dye serves as a sink, reacting with acetaldehyde to form a Schiff base which is intensely red (22). This reaction would be expected to shift the equilibrium for alcohol hydrogenase toward aldehyde production. The red product was still quite soluble and diffused into the surrounding agar.

                  TABLE 1                                                          ______________________________________                                         Plasmids and strains used                                                      Strain or    Relevant         Source or                                        plasmid      genotype         reference                                        ______________________________________                                         Z. mobilis CP4                                                                              Prototroph       29                                               E. coli                                                                        TC4          recA lacY        4                                                DH5α   lacI.sup.q ΔlacZM15 recA                                                                  BRL.sup.a                                        JM101        lacI.sup.q ΔlacZM15                                                                       BRL                                              Plasmids                                                                       pUC18        bla lacI`Z`.sup.b                                                                               40                                               pLOI193      cat mob.sup.+    8                                                pLOI284      bla adhB         This study                                       pLOI285      cat adhB         This study                                       pLOI286      bla adhB         This study                                       pLOI287      bla adhB         This study                                       ______________________________________                                          .sup.a Bethesda Research Laboratories.                                         .sup.b Incomplete lacI and incomplete lacZ.                              

Positive colonies were best visualized in low numbers (less than 20 positive colonies per plate containing 100 to 300 total colonies) after overnight incubation at 37° C. (with or without added antibiotics). Such colonies completely colored the plates after prolonged incubation. Active colonies appeared intensely red, with negative colonies ranging from white to medium shades of red, depending upon the level of background color in the plate, a function of indicator plate age and storage conditions. Fresh indicator plates were prepared no more than 2 days before use.

Colonies selected by antibiotic resistance were tested for alcohol dehydrogenase activity by using sterile toothpicks or applicator sticks to transfer cells to gridded aldehyde indicator plates. Positive clones were detected after 20 min to 2 h at 37° C. (FIG. 1A). During overnight or longer incubation, negative colonies were surrounded by a halo which was lighter than the background plate (FIG. 1B), presumably due to the consumption of aldehydes by the aldehyde dehydrogenase activity known to be present in E. coli (6). The relatively low levels of native alcohol dehydrogenase in E. coli (5) may contribute to the modest color development in negative clones during incubation.

The addition of glucose and other readily fermentable sugar resulted in sufficient aldehyde production to darken plates and produce red colonies with little diffusion of colored product. However, modifications of this plate assay can be used to screen for pyruvate decarboxylase activity (more intensely red with diffusion of pigment) by using glucose for growth or pyruvate instead of ethanol as a substrate.

Cloning of the adhB gene. Chromosomal DNA was isolated from Z. mobilis as described previously (4). This DNA was partially digested with Sau3A, and fragments of 5 to 7 kilobases (kb) were isolated by using a sucrose gradient (7). A Z. mobilis library was constructed by ligating these fragments into the BamHI site of the tetracycline resistance gene of pLOI193 (8). Ligated preparations were transformed into E. coli TC4 with selection for chloramphenicol resistance. Three ligations were used to construct the library. Each ligation yielded approximately 10,000 colonies, of which over 50% contained an insert. These original plates were scraped and transferred into centrifuge tubes, and the plasmid DNA was isolated by using the alkaline sodium dodecyl sulfate method (25). This DNA served as an amplified library and gene bank from which alcohol dehydrogenase clones were isolated as new transformants of strain TC4 on aldehyde indicator plates containing chloramphenicol. Alcohol dehydrogenase activity was confirmed by direct spectrophotometric assay by measuring the acetaldehyde-dependent oxidation of NADH.

Subclones were prepared by the ligation of fragments from purified plasmids into the vector pUC18 (40). These ligations were transformed into E. coli DH5α (Table 1) and plated on Luria agar containing ampicillin and X-Gal. Clones containing inserts (negative for β-galactosidase) were screened for alcohol dehydrogenase activity by picking onto aldehyde indicator plates. These were back-transformed into strain TC4 for further study.

Assay of alcohol dehydrogenase. Alcohol dehydrogenase activity was determined by measuring the acetaldehyde-dependent oxidation of NADH or by measuring the alcohol-dependent reduction of NAD⁺ by using ethanol or butanol (28). Cell suspensions in buffer (0.1M Tris maleate, 1 mM dithiothreitol [pH 6.5]) were disrupted by the addition of lysozyme (0.5 mg/ml) and vigorous mixing with chloroform (0.025 ml of chloroform added to 0.2 ml of cell suspension). Activities are expressed as micromoles per minute per milligram of total cell protein. Cell protein was determined by using the method of Lowry et al. (23) with bovine serum albumin as a standard.

Acrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described previously (9). For zymograms of alcohol dehydrogenase isozymes, cells were disrupted with lysozyme and detergent (28) and separated on 7% polyacrylamide gels with a 3% stacking gel essentially as previously described (38). Alcohol dehydrogenase activity was visualized by using phenazine methosulfate and nitrotetrazolium blue (15).

DNA sequence analysis. The gene encoding alcohol dehydrogenase II from Z. mobilis was sequenced by the dideoxy method of Sanger et al. (35) as described previously (8). The entire sequence of the Z. mobilis DNA insert of pLOI284 was determined for both strands. Staggered deletions of about 175 base pairs (bp) each were prepared by opening the plasmid at one end of the insert, followed by timed digestions with the nuclease BAL 31 (25). The deletion fragments were digested with a second restriction enzyme, which cut at the opposite end of the insert, and subcloned into bacteriophage M13mp18 or M13mp19. In addition, the 402-bp DraI fragment of pLOI287 (immediately upstream of the sequence present in pLOI284) was sequenced in both directions by cloning into M13mp18. The sequence data were analyzed by using the programs of Pustell and Kafatos (32), obtained from International Biotechnologies, Inc., Beverly, Mass.

Analysis of transcriptional initiation sites. The 5' termini of the transcripts were mapped by the primer extension method (31). RNA isolation and mapping experiments were performed as described previously (9). The primer used for these experiments was synthesized by using an Autogen 500 oligonucleotide synthesizer (Millipore Corp., Bedford, Mass.) and was complementary to the noncoding strand of the adhB gene (3'-GCAGTTGCTTTACCC-5'), spanning bp 206 (3') to 220 (5'). The cDNA prepared in these experiments was compared to a sequence ladder generated with the same primer and the appropriate M13 clone containing the homologous DNA fragment.

Enzymes and chemicals. Restriction enzymes were obtained from International Biotechnologies. Avian myeloblastosis virus reverse transcriptase and the Klenow fragment of DNA polymerase I were obtained from Bethesda Research Laboratories, Inc., Gaithersburg, Md. Dideoxy- and deoxynucleotides were purchased from Pharmacia, Inc., Piscataway, N.J. Biochemicals, pararosaniline, and sodium bisulfite were purchased from Sigma Chemical Co., St. Louis, Mo. Radioactive compounds were obtained from New England Nuclear Corp., Boston, Mass.

Results

Cloning of the alcohol dehydrogenase II gene from Z. mobilis. A library of Z. mobilis genomic DNA in the broad host range vector pLOI193 (8) was transformed into E. coli TC4 and plated on aldehyde indicator plates containing chloramphenicol. More than 20,000 colonies were screened, and three positive clones were found, similar to those shown in FIG. 1. The presence of alcohol dehydrogenase was confirmed in the three clones by direct spectrophotometric assay. These clones appeared to be identical in that they contained inserts of 5.6 kb and were digested into identical fragments by EcoRV and DraI. One of these clones was designated pLOI285 and retained for further study.

The 5.6-kb Z. mobilis DNA fragment was mapped by using a variety of restriction endonucleases. The first subclone retaining a functional adhB gene was made by digestion of pLOI285 with EcoRV, followed by ligation of the fragments into the SmaI site of pUC18. This subclone, designated pLOI287, contained two EcoRV fragments (2.2 and 1.2 kb long). Both of these fragments were required to retain alcohol dehydrogenase activity. A subclone of pLOI287 was constructed by digestion with BstEII and EcoRI, followed by conversion of the staggered cuts to blunt ends by using the Klenow fragment of DNA polymerase I and self-ligation. This plasmid, designated pLOI286, was further shortened by subcloning the DraI fragments of the Z. mobilis insert DNA into the SmaI site of pUC18. This final construction was designated pLOI284 and contained the smallest pieces of Z. mobilis DNA which retained a functional alcohol dehydrogenase. It contained the two DraI fragments from pLOI286, 0.12 and 1.23 kb long, and retained a single, internal DraI site.

Sequence of the adhB gene from Z. mobilis. The sequence of the gene encoding alcohol dehydrogenase II in Z. mobilis is presented in FIG. 2. A single open reading frame of 1,149 bp was identified, corresponding to 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,141. The N-terminal amino acid sequence of Z. mobilis for this protein has been previously reported (28) and is identical to that predicted by our sequence through the first 36 amino acids. The sequences differ at amino acid 37, which was reported by Neale et al. (28) to be a glycine but is a serine in our sequence and may represent a strain variation (single base change). Amino acids in positions 38 through 49 are in agreement with those determined by peptide sequencing (28). The predicted amino acid composition from our sequence is in excellent agreement with that reported by Wills et al. (39).

The coding region of adhB is preceeded by the sequence GAGG, a probable ribosome-binding site (10, 21) located 8 bp upstream from the start codon. This gene ends with a single stop codon, TAA. A 13-bp palindrome which resembles a procaryotic terminator (30, 34) is located 50 bp downstream from this stop codon. We confirmed that the palidrome acts as a terminator by inserting a promoterless tetracycline gene downstream, a construction which did not confer resistance to tetracycline. The sequence upstream from the ribosome-binding site of adhB is extremely A+T rich and contains several potential -10 and -35 regions, although most of these do not show the proper spacing to be considered homologous to the promoter consensus sequence described for E. coli (17, 34).

Expression of the adhB gene in E. coli. Z. mobilis contains two isozymes for alcohol dehydrogenase which can be readily distinguished by their ability to oxidize butanol and by their rates of migration on zymograms (20, 28, 39). By using these criteria, the activity produced in E. coli by clones containing pLOI286 and pLOI284 was identified as alcohol dehydrogenase II from Z. mobilis. Cell extracts rapidly oxidized ethanol but failed to oxidize butanol. On native gels, this activity migrated adjacent to alcohol dehydrogenase II from extracts of Z. mobilis (data not shown). Sodium dodecyl sulfate-polyacrylamide gels of extracts from Z. mobilis CP4 and E. coli TC4 containing pLOI284 were also examined. Strain TC4 containing pLOI284 exhibited an increase in protein in the region corresponding to 38,000 to 40,000 daltons, although this band was not cleanly resolved from native proteins (data not shown).

During the construction of pLOI284, only a single orientation of fragments in pUC18 was obtained, suggesting that the expression of adhB may be dependent upon the lac promoter in pUC18 for transcription. If a specific activity for pure enzyme of 170 U (28) is assumed, alcohol dehydrogenase II from Z. mobilis can be calculated to represent 0.25% of the total cellular protein in E. coli grown without glucose (Table 2). This activity was roughly halved by growth in the presence of glucose (Table 2), consistent with the adhB gene in pLOI284 being transcribed under the control of the lac promoter (12). In contrast, the level of activity expressed by strain TC4 containing pLOI286 was much lower and exhibited a smaller decline during growth with glucose.

Mapping of transcriptional initiation of adhB. The sites of transcriptional initiation were identified by primer extension analysis by using mRNA isolated from Z. mobilis CP4 during the logarithmic growth phase (FIG. 3A). Two prominent bands were evident and of equal intensity, indicating that transcription of adhB in Z. mobilis was initiated from two different promoters. The second of these, designated P2, was assigned as bp 1 (a thymidine) in FIG. 2 and is 177 bp upstream of the translational start codon. The other initiation site, designated P1, is also a thymidine and is located 277 bp upstream of the translational start codon.

Primer extension experiments using mRNA from E. coli TC4 containing pLOI286 (carrying the entire promoter region of adhB) indicated a weak site of initiation directly adjacent to the thymidine used by Z. mobilis at P2 (FIG. 3A; visible with longer exposure). Strain TC4 did not appear to use P1. A similar experiment with strain TC4 carrying pLOI284, which contains a shortened adhB lacking both Z. mobilis promoters, exhibited a single site of transcriptional initiation from the lac promoter supplied by pUC18 (FIG. 3B).

Each of the tandem promoters used by Z. mobilis is similar to the consensus promoter sequence for E. coli (17, 34), although they differ from one another. The -10 region of P2 has the sequence CATATT, with four of six bases in common with the consensus sequence (17, 34). The -10 region of P1 contains the sequence TAGAAA, also with four of six bases matching the consensus sequence. The -35 regions of P2 and P1 are nearly homologous, with the sequences TTGATC and TTGCTC, respectively. The first shares four of six bases in common with the -35 consensus sequence, and the latter shares three of six bases. Both of

                  TABLE 2                                                          ______________________________________                                         Specific activity of alcohol dehydrogenase clones                                                   Alcohol dehydrogenase                                                          (μmol/min per mg of                                    Plasmid.sup.a                                                                              Glucose.sup.b                                                                           total cell protein).sup.c                                 ______________________________________                                         pLOI284     +        0.18                                                                  -        0.43                                                      pLOI286     +        0.08                                                                  -        0.10                                                      No plasmid  +        0.04                                                                  -        0.05                                                      ______________________________________                                          .sup.a Plasmids contained in E. coli TC4.                                      .sup.b Cells were grown in Luria broth in the presence or absence of           glucose (20 g/liter).                                                          .sup.c Measured by acetaldehydedependent oxidation of NADH.              

these promoters have the proper spacing of 17±1 bp between the -10 and -35 regions (17, 34).

Discussion

The adhB gene from Z. mobilis was transcribed from tandem promoters. This is not unexpected for an important, highly regulated gene and is not uncommon in other procaryotes (13, 26). However, Z. mobilis is obligately fermentative and requires alcohol dehydrogenase for the oxidation of NADH under all growth conditions. The Z. mobilis adhB promoter structure appears unusual in two ways. First, both of the adhB promoters were used by Z. mobilis during log-phase growth. Normally, tandem promoters are differentially expressed (3, 33). Tandem promoters of the adh gene from Drosophila melanogaster are expressed separately, at different stages of development (1). A second unusual feature of the tandem Z. mobilis promoters is the uncharacteristically long, untranslated 5'-mRNA leaders. The significance of such leaders is unknown.

The two adhB promoters of Z. mobilis were not used efficiently for expression in E. coli, although both share homology with the consensus promoter sequence of E. coli (17, 34). The P2 promoter of adhB was used to a limited extent, and the P1 promoter appeared to be unused by E. coli. The -35 regions of the two adhB promoters are nearly identical. It is likely that the differences between the two adhB promoters in the -10 region are important for utilization by E. coli and for the regulation of alcohol dehydrogenase II activity in Z. mobilis. High levels of alcohol dehydrogenase II are present in this obligately ethanologenic bacterium, although the proportions of the two isozymes have been reported to vary with growth conditions and at different stages of batch fermentation (20). In E. coli, transcription from the lac promoter was blocked in pLOI286 but not in pLOI284, although no palindromic sequences are evident in the additional upstream region of pLOI286 which could serve as transcriptional terminators.

The other enzyme required by Z. mobilis for the regeneration of NAD⁺, i.e., pyruvate decarboxylase, has also been cloned (2) and sequenced (9). The promoter for pdc is unused by E. coli but serves to express this gene at high levels during fermentation in Z. mobilis. No sequence homology was evident between the promoters of pdc and adhB (data not shown). The predicted peptide sequence of Z. mobilis alcohol dehydrogenase II exhibited no appreciable homology to reported sequences for alcohol dehydrogenases from maize, yeasts, mammals, or D. melanogaster. A small region of possible homology in the amino end with yeasts has been described previously (28). The apparent lack of homology is consistent with the unusual nature of the Z. mobilis alcohol dehydrogenase II enzyme, reported to be the only alcohol dehydrogenase to contain iron rather than zinc (36).

The transcription of both adhB (pLOI284) and pdc (pLOI276; 9) was initiated from the lac promoter (derivatives of pUC18) in E. coli TC4. However, the level of pyruvate decarboxylase produced in strain TC4 was eightfold higher than the level of alcohol dehydrogenase II when compared as a percentage of total cellular protein. Such differences in levels of enzyme may reflect differences in mRNA stability, protein stability, or efficiency of translation (19). In Z. mobilis, alcohol dehydrogenase (both isozymes) also represents a smaller fraction of cellular protein than does pyruvate decarboxylase, although both activities are abundant (18).

The protein encoded by the gene of the invention can be used to convert ethanol, a low value product of fermentation, to acetaldehyde, a product of higher value. This conversion can be done by contacting organisms expressing the subject protein with ethanol in the presence of air. This is shown in FIG. 1. Acetaldehyde is a known flavoring agent in foods and beverages.

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We claim:
 1. An isolated DNA fragment encoding bacterial alcohol dehydrogenase which has an amino acid sequence as shown in FIG.
 2. 